Investigation of putative regulatory loci relevant to the pathogenesis of psychiatric illness

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Abstract

The genetic contribution to the aetiology of psychiatric illness is well-established; however,
few variants that alter the encoded protein have been irrefutably identified as causative,
leading to the hypothesis that variants affecting gene regulation may play a pathogenic role.
This thesis focuses on two genes, Neuregulin 1 (NRG1) and Disrupted in Schizophrenia 1
(DISC1), for which there is strong genetic evidence for involvement in psychiatric illness, as
well as evidence for altered expression in patients.
Association analysis was carried out to assess the involvement of six intronic NRG1 single
nucleotide polymorphisms (SNPs) in schizophrenia and bipolar disorder in two independent
samples from the Scottish (Scottish 2; n = 307 control subjects, 303 schizophrenic patients,
and 239 bipolar disorder patients and German populations (n = 397 control subjects, 396
schizophrenic patients, and 400 bipolar disorder patients). These SNPs form two haplotypes,
one encompassing the 5’ and promoter region of the gene and the other located at the 3’ end
of the gene, that were previously associated with schizophrenia and bipolar disorder in a
Scottish sample (Scottish 1). The location of these haplotypes, together with the prior
evidence for altered NRG1 expression in schizophrenia, suggested the potential involvement
of regulatory variants. On combining the Scottish 1 and Scottish 2 samples (combined n =
765 control subjects, 682 schizophrenic patients and 601 bipolar disorder patients), a two-
SNP haplotype spanning both coding and non-coding regions in the 3’ region was associated
with schizophrenia (p = 0.0037, OR=1.3, 95% CI: 1.1-1.6) and the combined schizophrenia
and bipolar disorder case group (p = 0.0080, OR=1.2, 95% CI: 1.1-1.5), with both these
associations remaining significant after permutation analysis (p = 0.022 and p = 0.044,
respectively).
To further understanding of how DISC1, a leading candidate gene for schizophrenia that has
also been implicated in other psychiatric disorders, is regulated the previously
uncharacterised promoter region was assessed both bioinformatically and in vitro using the
dual luciferase reporter assay. The region was found to lack canonical promoter motifs but to
contain a CpG island, consistent with DISC1’s ubiquitous pattern of expression. A region
located 300bp to -177bp relative to the transcription start site (TSS) was identified as
contributing positively to DISC1 promoter activity, whilst a region -982bp to -301bp relative
to the TSS was found to confer a repressive effect. FOXP2, a transcription factor which is
mutated in a rare speech and language disorder and implicated in autism pathogenesis, was
found to repress transcription from the DISC1 promoter. Two pathogenic FOXP2 point
mutations reduced this transcriptional repression. Preliminary evidence for a bi-directional
regulatory relationship between DISC1 and FOXP2 was observed: a mouse model of
schizophrenia that carries a Disc1 L100P amino acid substitution and shows altered
developmental Disc1 expression was also found to show altered developmental expression of
Foxp2.
These results further understanding of two genes whose altered expression might contribute
to the pathogenesis of psychiatric illness.